Gene name - erect wing
Cytological map position - 1A8
Function - Transcription factor
Symbol - ewg
Genetic map position - 1-0.0.
Classification - novel
Cellular location - nuclear
|Recent literature||Rai, M., Katti, P. and Nongthomba, U. (2016). Spatio-temporal coordination of cell cycle exit, fusion and differentiation of adult muscle precursors by Drosophila Erect wing (Ewg). Mech Dev [Epub ahead of print]. PubMed ID: 27039019
The mechanisms of cell cycle exit by myoblasts during skeletal muscle development are poorly understood. Cell cycle arrest is known to be a prerequisite for myoblast fusion and subsequent differentiation. Despite tremendous knowledge on myoblast fusion and differentiation, tissue-specific factors that spatio-temporally regulate the cell cycle exit are not well known. This study shows that the transcriptional factor/co-activator "Erect wing" (Ewg) synchronises myoblast cell cycle exit with that of the fusion process. Ewg-null myoblasts show delayed temporal development of dorsal longitudinal muscles (DLMs), a group of indirect flight muscles (IFMs), which culminates to abnormal and asymmetric muscle pattern. A role for Ewg in cell cycle exit at G1/S stage is also shown. Reducing Cyclin E dose in Ewg-null mutant rescues the lack of IFMs and flight ability. Thus, Ewg repression of Cyclin E expression is required for the arrest of myoblast proliferation and initiation of myoblast fusion and terminal differentiation.
EWG is a novel transcription factor with homology to DNA binding proteins found in organisms as diverse as sea urchins and mammals. Mutants exhibit both neural defects (apparent during embryonic development), and muscle defects, that are noticeable during the pupal phase (DeSimone, 1995).
Each type of defect, neural or muscle, has its own developmental basis. The neural defect is due to expression of ewg in neurons, while the muscle defect is due to expression of ewg in myoblasts. In fact, ewg is the first regulatory gene to be identified in Drosophila that is expressed in imaginal myoblasts. Before discussing the muscle defects, the embryonic origin of indirect flight muscles will be examined.
Myoblasts that contribute to the indirect flight muscles (IFMs) are derived from the embryonic mesoderm and attach to imaginal discs and nerves during larval development. At the onset of metamorphosis, these myoblasts migrate over the developing adult epidermis and fuse, forming the adult muscles. One group of indirect flight muscles, the dorsal longitudinal muscles (DLMs) uses modified larval muscles as templates for their development, while another group of very similar IFMs, the dorsoventral muscles (DVMs) appears to develop by the de novo fusion of myoblasts. The innervation of the IFMs develops from the modification of larval nerves. Neurons that innervate larval muscles withdraw their termini at the onset of metamorphosis, undergo specific modifications, and send out processes that grow over the developing IFMs (DeSimone, 1995 and references).
EWG expression in myoblasts is not detected during the third larval instar, however, at the same stage, EWG is detected in all larval neurons. After the onset of metamorphosis, myoblasts associated with the wing imaginal discs migrate over the developing adult epidermis. At this stage, and until 10 hours after pupal formation (APF) EWG protein is not detected in the myoblasts. At 10 hours APF, EWG protein is detected in a small population of cells in the region where DLMs are known to develop. These EWG-positive cells overlie the larval templates that are used for the development of DLMs. By 13 hours, the staining is stronger and more cells are labeled, while by 16 hours, the alignment of labeled cells along the surface of the larval templates is a noticeable feature. EWG expression is also seen in myoblasts that will contribute to the DVMS, in the progenitors of the jump muscles and their developing fibers. The first signs of defects in IMFs in ewg mutants are observed at about 18 hours APF when degeneration of the ventral-most DLMs becomes apparent. A little later, at 20 hours, the DLMs have completely degenerated (DeSimone, 1995).
To test whether neural or myoblast expression of ewg accounts for the defect in flight muscles, ewg was expressed in neurons of ewg mutants using an elav promoter. Neural expression of ewg rescues the embryonic lethality phenotype of ewg mutation but fails to rescue the IFM defects (DeSimone, 1995) It is concluded that neural expression alone is insufficient to rescue the muscle defect.
When a heat shock ewg expression vector is introduced in ewg mutant myoblast null strains, partial restoration of muscles is obtained. It is concluded that ubiquitous ewg expression allows for myogenesis, implying that muscle expression, but not neural expression (see above) cures the muscle defect in ewg mutants. This partial rescue results in animals with three fused DLMs instead of the normal six. The innervation to each of the three 'unsplit' muscles, which do not degenerate, resembles a composite of that normally seen over the pairs of muscles that will form in the wild type. Upon expression of EWG in the developing muscles, the axons branch in a manner similar to the wild type, suggesting that at least some cues for axon branching must come from the muscle targets. In neural ewg expression, innervation prior to muscle degeneration is only partial, that is to say, innervation is incomplete. Thus at least some aspect of innervation is directed by expression of ewg in muscle (DeSimone, 1995).
Formation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals. This study shows that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach it was demonstrated that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis it was demonstrated that EWG genetically interacts with Wingless and Notch, and also with TGF-β and AP-1 pathways in the regulation of synaptic growth. These results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network (Haussmann, 2008).
Several pathways have been identified that stimulate synaptic growth at NMJs of Drosophila larvae (Wnt/Wingless, TGF-β/BMP and jun kinase). Overexpression of AP-1 and mutants in regulatory genes involved in Wnt/Wingless and TGF-β/BMP pathways (spinster, highwire, shaggy and the proteasome) can increase bouton numbers, suggesting that synaptic growth is regulated through the balance of stimulatory and restrictive signals. This study has identified such a restrictive role for the transcription factor EWG and, through the analysis of EWG-regulated genes, for the N pathway in the regulation of synaptic growth. Using genetic mosaics, it was further demonstrated that EWG's role in synaptic growth regulation is cell-autonomous, suggesting that the transcriptional regulator EWG mediates this restrictive effect through the alteration of transcription pre-synaptically (Haussmann, 2008).
Analysis of genes differentially expressed in ewgl1mutants revealed a rather unexpected set of genes involved in synaptic growth regulation, besides an expected number of metabolic genes due to homology of EWG to human NRF-1. Most genes that could account for the phenotype of ewgl1mutants, and that are thus expressed in the nervous system, are involved in transcriptional and post-transcriptional regulation of gene expression. Although changes of transcript levels in ewgl1mutants were mostly moderate, their significance was validated through mRNA profiling with rescued ewgl1mutants under the same conditions of RNA preparation and microarray hybridization. In addition, differences in gene expression in ewgl1mutants were validated using quantitative RT-PCR and biochemical assays with regard to predicted changes in glycogen levels based on differential regulation of genes involved in gluconeogenesis. Furthermore, genetic interaction experiments in double mutants with increased bouton numbers support that these co-regulated genes are functionally connected in regulating synaptic growth (Haussmann, 2008).
The group of neuronal genes among those differentially regulated in ewgl1mutants that have been demonstrated to have roles in synaptic growth or could account for it, is remarkably small. In particular, from the large number of cell adhesion molecules and cytoskeletal proteins present in the Drosophila genome only a handful is differentially regulated. Similar results have also been obtained in response to JNK and AP-1 signaling. These results are in contrast to changes in gene expression induced by acute or chronically enhanced neuronal activity in Drosophila seizure mutants, which also result in synaptic overgrowth. Here, the vast majority of differentially regulated genes are for cell adhesion molecules and cytoskeletal proteins or their regulators, and genes involved in synaptic transmission and neuronal excitability; transcriptional or post-transcriptional regulators comprise only a minor portion. These differences could be explained by separate pathways regulating growth independent of neuronal activity (Haussmann, 2008).
Particularly striking is the large number of genes involved in RNA processing among genes differentially expressed in ewgl1mutants. Although local regulation of gene expression is required in growth cones of navigating axons, a prominent role for pre-synaptic regulation of gene expression at the RNA level is only just emerging, but is a hallmark of post-synaptic plasticity. Several RNA binding proteins have been implicated in memory storage . osk and CPSF (cleavage and polyadenylation specificity factor) are among the genes differentially regulated in ewgl1mutants. Other genes involved in RNA processing differentially regulated in ewgl1mutants comprise the whole spectrum of regulation at the post-transcriptional level, from nuclear organization (otefin), alternative pre-mRNA processing (Pinin, CPSF, Rox8) and export/import (Segregetion distorter, Nxf2, CG11092, Karyopherin, Transportin) to transport, localization and translation (oskar, swallow, ribosomal protein genes S5 and Rpl24), and likely also include the regulation of mRNA stability (Rox8) (Haussmann, 2008).
An intriguing connection between ewg and signaling pathways involved in regulating synaptic growth is indicated by differentially regulated components of the Wg and N pathways (gro and Hairless) in ewgl1mutants. Consistent with a role of the co-repressor gro in Wg and N mediated transcriptional regulation of synaptic growth, Wg and N signaling pathways do not operate independently of ewg in genetic interaction experiments. The transcriptional regulatory networks of EWG, Wg and N seem to be highly interwoven. Overexpression of pan, the transcriptional mediator of canonical Wg signaling, which is repressed by gro, does not lead to a further expansion of synaptic growth in ewg mutants, suggesting a requirement for ewg-regulated genes. This effect could be mediated by deregulated N signaling, which is also repressed by gro, but antagonistic to Wg in synaptic growth. Thus, removal of gro, as in ewg, will relieve the repressive effect of N and antagonize the stimulatory effect of pan. In the complementary situation, removal of N increased bouton numbers further in the absence of EWG, which is consistent with an increase in Wg signaling as a result of down-regulated gro in ewg mutants. Antagonism between N and Wg pathways has also been found in wing discs, where N inhibits armadillo (arm), the transcriptional co-activator of canonical Wg signaling. Intriguingly, gro has also been found to be a target of receptor tyrosine kinase signaling and, thus, can combine additional pathways with N and Wg signaling. In addition to transcriptional hierarchies, chromatin remodeling has also been implicated in synaptic plasticity. Strikingly, CG6297, a Drosophila homologue of the histone deacetylase RPD3, is differentially expressed in ewgl1mutants and physically interacts with gro (Haussmann, 2008).
How ewg exerts its effect on TGF-β signaling is less clear. A prominent regulatory step in this pathway is the regulated degradation of the SMAD co-factor Medea by Highwire. Several genes involved in regulating protein stability are differentially down-regulated in ewg mutants (CG6759, CG3431, CG4973, CG7288, CG3455, CG9327 and CG9556). Lower expression levels of these genes might interfere with stabilization of Medea and explain why the effect of activated TGF-β signaling is not additive in the absence of EWG (tkvA GOF ewg LOF). Bouton numbers in wit null mutants are marginally increased in the absence of EWG, suggesting further that genes regulated by SMADs are involved in mediating synaptic overgrowth in ewgl1mutants. Potentially, ewg could also regulate TGF-β signaling through the endosomal pathway involving spinster and/or spichthyin (Haussmann, 2008).
Functionally related genes have been shown to be co-regulated, suggesting additional ELAV targets in EWG-regulated gene networks. Indeed, ELAV negatively regulates alternative splicing of the penultimate exon in armadillo (arm). Exclusion of this exon, which truncates the carboxyl terminus of arm, reduces Wg signal transduction, which is in agreement with ewg's antagonistic role relative to Wg signaling. Another known ELAV target gene is neuroglian (nrg), where a role in synapse formation has recently been demonstrated in the giant fiber system. Taken together, the establishment of a gene network regulated by EWG will now serve as valuable tool to identify further ELAV regulated modules that shape the synapse (Haussmann, 2008).
The transcription factor EWG is a major target of the RNA binding protein ELAV, which regulates EWG protein expression via a splicing mechanism. EWG is required pre-synaptically and cell-autonomously at third instar neuromuscular junctions to restrict synaptic growth, demonstrating that restrictive activities at gene expression levels are also required for synaptic growth regulation. EWG mediates regulation of synaptic growth primarily by increasing transcript levels of genes involved in transcriptional and post-transcriptional regulation of gene expression. Genes at the end of the gene expression hierarchy (effector genes) represent only a minor portion of EWG-regulated genes. Since analysis of mutants in genes differentially regulated in ewgl1mutants revealed that these genes are involved in both stimulatory and restrictive pathways of synaptic growth, and since ewg genetically interacts with a number of signaling pathways (Wingless, Notch, TGF-β and AP-1), the results suggest that synaptic growth in Drosophila is regulated by the interplay of multiple signaling pathways rather than through independent pathways (Haussmann, 2008).
Bases in 5' UTR - 1962 and 424
Bases in 3' UTR - 748
EWG and S. purpuratus P3P2 are highly homologous. The two proteins show striking homology in the 225 amino acid region between residues 123 and 349 of EWG. In this region the proteins are 71% identical. Overall, the proteins show 48% identity. EWG contains long stretches of basic or acidic amino acids. In EWG, a large, highly basic region extends from residue 86 to residue 124 and contains 15 aspartite and glutamate residues. There is a large basic region extending from residue 147 to residue 219. The amino-terminal end of the large basic domain resembles nuclear localization signals. The region between the large acidic and basic domains is very rich in alanine residues and is predicted to form a helix-turn-helix DNA binding motif. 87 residues near the middle of the protein contain 19 glutamine and 20 alanine residues (DeSimone, 1993).
Genetic studies of the Drosophila erect wing (ewg) gene have revealed that ewg has an essential function in the embryonic nervous system and is required for the specification of certain muscle cells. Ewg is a site-specific transcriptional activator, and evolutionarily conserved regions of Ewg contribute both positively and negatively to transcriptional activity. Using gel mobility shift assays, it has been shown that an Ewg dimer binds specifically to DNA. In transfection assays, Ewg activates expression of a reporter gene bearing specific binding sites. Analysis of deletion mutants and fusions of Ewg to the Gal4 DNA binding domain has identified a transcriptional activation domain in the C terminus of Ewg. Deletion analysis has also revealed a novel inhibitory region in the N terminus of Ewg. Strikingly, both the activation domain and the inhibitory region are conserved in Ewg homologs including human nuclear respiratory factor 1 (NRF-1) and the sea urchin P3A2 protein. The strong conservation of elements that determine transcriptional activity suggests that the Ewg, NRF-1, and P3A2 families of proteins shares common mechanisms of action and have maintained common functions across evolution (Fazio, 2001).
Analysis of Ewg deletion derivatives has revealed functional roles in transcriptional regulation for each of the regions of Ewg that are conserved in the human NRF-1 and sea urchin P3A2 homologs with the exception of residues 543-564. In contrast, these studies failed to reveal a critical role for the nonconserved regions of Ewg. Because of this strong correlation of sequence conservation with function, it seems likely that conserved residues 543-564 contribute to Ewg activity in certain physiological contexts not reproduced in these assays. Consistent with the proposal that the highly conserved region of Ewg from residues 146-343 participates in nuclear localization, DNA binding, and dimerization, it was found that deletion of the sequences C-terminal to amino acid 350 does not impair DNA binding or dimerization, and deletion of the sequences N-terminal to position 144 does not reduce, but rather stimulates, site-specific activation. The minimal activation domain has been mapped to residues 564-654, which includes a highly conserved core, residues 631-654, necessary for activation. Unexpectedly, a conserved region in the N terminus was found to inhibit activation by Ewg. The identification of evolutionarily conserved regions that both positively and negatively influence transcriptional activation by Ewg raises the possibility that the activity of Ewg and its homologs may be regulated by common mechanisms dependent upon cell type or promoter context (Fazio, 2001).
A wide variety of unrelated amino acid sequences has been shown to function as activation domains; thus it is particularly striking that the Ewg activation domain is so highly conserved. An analysis of NRF-1 deletions has revealed a major role for NRF-1 residues 449-477 in activation that corresponds well to the highly conserved core that is critical for the function of the Ewg activation domain. In the case of NRF-1, the mutation of hydrophobic residues in the activation domain also significantly reduces activation, although mutation of multiple glutamines in the activation domain has no effect. NRF-1 has been shown to bind the coactivator PGC-1 through its DNA binding domain, but no targets of the NRF-1 activation domain have been identified. The high sequence conservation of the activation domain across evolution strongly suggests that Ewg and NRF-1 activate transcription by a common mechanism. The core activation domain is well conserved in the sea urchin P3A2 homolog, which has been shown to repress transcription. It is interesting to note that in P3A2 there is alanine at the position equivalent to Val-641 in Ewg because a double mutant in which alanine replaces both Val-639 and Val-641 is severely impaired for activation. Thus, it is possible that P3A2 does not activate transcription because of changes in the region homologous to the activation domain or, alternatively, that this function may be conserved and P3A2 may function both as an activator and as a repressor (Fazio, 2001).
Analysis of Ewg deletion derivatives reveals the presence of a novel conserved inhibitory domain in the amino terminus. Deletion of residues 87-144 results in a 55-fold increase in activation. Modulation of transcriptional activation by Ewg may be critical because overexpression of Ewg in Drosophila, particularly outside the nervous system, is lethal. Previous studies with NRF-1 found that deletion of the N terminus (Delta77) results in decreased DNA binding, which in the case of NRF-1 is due to a defect in dimerization. It was therefore surprising that deletion of the N terminus of Ewg results in increased activation, particularly because deletion of Ewg residues 1-144 was found to reduce DNA binding activity in vitro. These results suggest that the N terminus of NRF-1 may also function to inhibit activation. Interestingly, phosphorylation of the N terminus of NRF-1 in response to extracellular signals has been shown to increase transcriptional activation by NRF-1 because of an increase in DNA binding (although not dimerization). The N terminus of NRF-1 has also been reported to interact with dynein light chains, although the functional significance of this is unknown. The conserved inhibitory region is rich in acidic residues, but the sequence provides no clues as to whether inhibition involves interactions in cis with other regions of Ewg or in trans with corepressor proteins. If the latter mechanism applies, it is possible that this amino-terminal domain contributes to transcriptional repression by P3A2. It will be interesting to determine whether the inhibitory activity of the Ewg N-terminus is modulated in response to extracellular signaling pathways or through interactions with other transcription factors at specific promoters (Fazio, 2001).
Expression of the 116-kDa form of Ewg is enriched in the Drosophila nervous system because of differential efficiency of splicing. Several other splice variants of Ewg mRNA have been described with the potential of encoding additional Ewg isoforms. The identification of functional domains in Ewg supports predictions of the functions of other forms of Ewg protein that may be expressed. All of the observed splice variants encode the N terminus, DNA binding, and dimerization domains. Alternative splicing of exon D (amino acids 386-540), which is not conserved between Ewg and NRF-1, has been observed in both neuron-rich heads and neuron-poor bodies. In analysis of Gal4 + Ewg fusions, residues encoded by exon D were not required for activity; therefore the data suggest that Ewg forms both containing and lacking exon D should function as transcriptional activators. Interestingly, the highly conserved region that is critical for function of the Ewg activation domain is present in a single small exon, exon H, which encodes amino acids 627-668. Thus, it is predicted that Ewg proteins derived from the observed splice variants lacking exon H would fail to activate transcription. Although transgenic studies have shown that the 116-kDa form of Ewg rescues development of the indirect flight muscle, the form of Ewg expressed in muscle has not been identified. It is of obvious interest to determine whether Ewg activity in the nervous system and in myoblasts requires the same functional domains (Fazio, 2001).
date revised: 15 August 2013
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